Activated Complement Components Research Articles

Accurate Visualization of C4d Complement Fragment in Immunohistochemistry by C-Terminal Linear Neoepitope-Specific Antibodies

C4d is the end degradation product of activated complement component C4b that appears during the early steps of the classical and lectin complement pathways. Within the primary sequence of C4d, there is a reactive thioester group that binds covalently to nearby surfaces, thus labeling the locations of complement activation. This feature makes C4d a target for immunohistochemical staining aimed to aid the diagnosis of, among others, the antibody-mediated rejection of transplanted organs, membranous glomerulonephritis, bullous pemphigoid, or inflammatory myopathies. However, the credibility of C4d immunostaining is debatable, as a high background in surrounding tissues and body fluids and diffused patterns of deposits in target structures are experienced with some of the available anti-C4d antibodies. Herein, we present an improved version of a rabbit anti-C4d antibody, originally raised against the C-terminal linear neoepitope of this complement fragment. Minor cross-reactivity with C4b and native C4 proteins, measured by ELISAs, as well as relatively low concentrations necessary for obtaining a specific signal in immunohistochemical analyses of formalin-fixed paraffin-embedded material, makes the improved antibody superior to commercially available rabbit monoclonal anti-C4d antibody SP91 dedicated to ex vivo diagnostics, as demonstrated by the staining of a panel of kidney transplant biopsies.

The complement system in neurodegenerative and inflammatory diseases of the central nervous system.

Neurodegenerative and neuroinflammatory diseases, including Alzheimer's disease, Parkinson's disease, and multiple sclerosis, affect millions of people globally. As aging is a major risk factor for neurodegenerative diseases, the continuous increase in the elderly population across Western societies is also associated with a rising prevalence of these debilitating conditions. The complement system, a crucial component of the innate immune response, has gained increasing attention for its multifaceted involvement in the normal development of the central nervous system (CNS) and the brain but also as a pathogenic driver in several neuroinflammatory disease states. Although complement is generally understood as a liver-derived and blood or interstitial fluid operative system protecting against bloodborne pathogens or threats, recent research, particularly on the role of complement in the healthy and diseased CNS, has demonstrated the importance of locally produced and activated complement components. Here, we provide a succinct overview over the known beneficial and pathological roles of complement in the CNS with focus on local sources of complement, including a discussion on the potential importance of the recently discovered intracellularly active complement system for CNS biology and on infection-triggered neurodegeneration.

Astrocyte-secreted C3 signaling impairs neuronal development and cognition in autoimmune diseases

Neuromyelitis optica (NMO) arises from primary astrocytopathy induced by autoantibodies targeting the astroglial protein aquaporin 4 (AQP4), leading to severe neurological sequelae such as vision loss, motor deficits, and cognitive decline. Mounting evidence has shown that dysregulated activation of complement components contributes to NMO pathogenesis. Complement C3 deficiency has been shown to protect against hippocampal neurodegeneration and cognitive decline in neurodegenerative disorders (e.g., Alzheimer's disease, AD) and autoimmune diseases (e.g., multiple sclerosis, MS). However, whether inhibiting the C3 signaling can ameliorate cognitive dysfunctions in NMO remains unclear. In this study, we found that the levels of C3a, a split product of C3, significantly correlate with cognitive impairment in our patient cohort. In response to the stimulation of AQP4 autoantibodies, astrocytes were activated to secrete complement C3, which inhibited the development of cultured neuronal dendritic arborization. NMO mouse models exhibited reduced adult hippocampal newborn neuronal dendritic and spine development, as well as impaired learning and memory functions, which could be rescued by decreasing C3 levels in astrocytes. Mechanistically, we found that C3a engaged with C3aR to impair neuronal development by dampening β-catenin signalling. Additionally, inhibition of the C3-C3aR-GSK3β/β-catenin cascade restored neuronal development and ameliorated cognitive impairments. Collectively, our results suggest a pivotal role of the activation of the C3-C3aR network in neuronal development and cognition through mediating astrocyte and adult-born neuron communication, which represents a potential therapeutic target for autoimmune-related cognitive impairment diseases.

O232: Enzymatic blood group conversion prevents complement activation in an ABO-incompatible model of human kidney transplantation

Abstract Introduction Recently, enzymatic blood group conversion has been used to convert human kidneys from blood group A to universal blood group O using bacterial enzymes from Flavonifractor plautii. However, little is known about the functional consequences of antigen removal when a treated kidney is transplanted in an ABO-incompatible (ABOi) recipient. Here, we investigated classical complement activation during ABOi conditions in enzyme treated (ABOe) vs control human type A kidneys. Method Three biological pairs of type A human kidneys rejected for transplantation and offered for research were used in this study. One kidney per pair was treated with 1mg/L of FpGalNAc deacetylase and FpGalactosaminidase during 6hrs of hypothermic machine perfusion while the contralateral kidney was perfused without enzymes. Both kidneys were subsequently perfused for 4hrs in ABOi conditions using normothermic machine perfusion (10% AB serum; mouse anti-A IgM titre 1:128; type O red blood cells). Tissue-bound components of classical complement activation were assessed in pre- and post-perfusion biopsies. Results Following ABOi perfusion, control kidneys showed peritubular deposition of the classical complement component C1qA and further complement activation components C4d and C5b-9. The staining directly co-localised with regions of anti-A staining. In ABOe kidneys, no anti-A staining was observed, with no C1qA, C4d, or C5b-9 staining found in post-perfusion biopsies. Conclusions We have shown for the first time that ABOe kidneys do not activate the classical complement pathway in ABOi conditions. This work shows the potential of the enzymatic blood group conversion to evade hyperacute antibody-mediated rejection during renal transplantation.

Elevated expression of complement factor I in lung cancer cells associates with shorter survival–Potentially via non-canonical mechanism

While numerous membrane-bound complement inhibitors protect the body's cells from innate immunity's autoaggression, soluble inhibitors like complement factor I (FI) are rarely produced outside the liver. Previously, we reported the expression of FI in non-small cell lung cancer (NSCLC) cell lines. Now, we assessed the content of FI in cancer biopsies from lung cancer patients and associated the results with clinicopathological characteristics and clinical outcomes. Immunohistochemical staining intensity did not correlate with age, smoking status, tumor size, stage, differentiation grade, and T cell infiltrates, but was associated with progression-free survival (PFS), overall survival (OS) and disease-specific survival (DSS). Multivariate Cox analysis of low vs. high FI content revealed HR 0.55, 95 % CI 0.32-0.95, p=0.031 for PFS, HR 0.51, 95 % CI 0.25-1.02, p=0.055 for OS, and HR 0.32, 95 % CI 0.12-0.84, p=0.021 for DSS. Unfavorable prognosis might stem from the non-canonical role of FI, as the staining pattern did not correlate with C4d - the product of FI-supported degradation of active complement component C4b. To elucidate that, we engineered three human NSCLC cell lines naturally expressing FI with CRISPR/Cas9 technology, and compared the transcriptome of FI-deficient and FI-sufficient clones in each cell line. RNA sequencing revealed differentially expressed genes engaged in intracellular signaling pathways controlling proliferation, apoptosis, and responsiveness to growth factors. Moreover, in vitro colony-formation assays showed that FI-deficient cells formed smaller foci than FI-sufficient NSCLC cells, but their size increased when purified FI protein was added to the medium. We postulate that a non-canonical activity of FI influences cellular physiology and contributes to the poor prognosis of lung cancer patients.

Acute neutrophilic vasculitis (leukocytoclasia) in 36 COVID-19 autopsy brains

BackgroundHypercytokinemia, the renin-angiotensin system, hypoxia, immune dysregulation, and vasculopathy with evidence of immune-related damage are implicated in brain morbidity in COVID-19 along with a wide variety of genomic and environmental influences. There is relatively little evidence of direct SARS-CoV-2 brain infection in COVID-19 patients.MethodsBrain histopathology of 36 consecutive autopsies of patients who were RT-PCR positive for SARS-CoV-2 was studied along with findings from contemporary and pre-pandemic historical control groups. Immunostaining for serum and blood cell proteins and for complement components was employed. Microcirculatory wall complement deposition in the COVID-19 cohort was compared to historical control cases. Comparisons also included other relevant clinicopathological and microcirculatory findings in the COVID-19 cohort and control groups.ResultsThe COVID-19 cohort and both the contemporary and historical control groups had the same rate of hypertension, diabetes mellitus, and obesity. The COVID-19 cohort had varying amounts of acute neutrophilic vasculitis with leukocytoclasia in the microcirculation of the brain in all cases. Prominent vascular neutrophilic transmural migration was found in several cases and 25 cases had acute perivasculitis. Paravascular microhemorrhages and petechial hemorrhages (small brain parenchymal hemorrhages) had a slight tendency to be more numerous in cohort cases that displayed less acute neutrophilic vasculitis. Tissue burden of acute neutrophilic vasculitis with leukocytoclasia was the same in control cases as a group, while it was significantly higher in COVID-19 cases. Both the tissue burden of acute neutrophilic vasculitis and the activation of complement components, including membrane attack complex, were significantly higher in microcirculatory channels in COVID-19 cohort brains than in historical controls.ConclusionsAcute neutrophilic vasculitis with leukocytoclasia, acute perivasculitis, and associated paravascular blood extravasation into brain parenchyma constitute the first phase of an immune-related, acute small-vessel inflammatory condition often termed type 3 hypersensitivity vasculitis or leukocytoclastic vasculitis. There is a higher tissue burden of acute neutrophilic vasculitis and an increased level of activated complement components in microcirculatory walls in COVID-19 cases than in pre-pandemic control cases. These findings are consistent with a more extensive small-vessel immune-related vasculitis in COVID-19 cases than in control cases. The pathway(s) and mechanism for these findings are speculative.

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Complement has long been recognized as a critical component of the innate immune system. It comprises proteins that play a central role in host defense against infection and in the modulation of antigen-specific immune and inflammatory responses. The complement system can be activated by three proteolytic cascades namely, the classical, the alternative, and the lectin pathways. The activation of complement components by all three pathways leads to the formation of a membrane attack complex (MAC). There are studies about complement system involvement in different ocular pathologies, like macular degeneration, glaucoma, diabetic retinopathy, and autoimmune uveitis. Dysregulation of the complement cascade has emerged as a key contributor to the pathophysiology of age-related macular degeneration and there has been a revolution in the treatment of the geographic type. The drug pegcetagoplan, which was approved by the FDA on February 17, 2023, is a C3 inhibitor that binds to C3 or C3b, disrupting further activation of the complement system. There are studies about the relationship between diabetic retinopathy and dysregulation of the complement system. Some authors found activation of C5a in the vitreous body, other authors found an increase of C9 and factor D in the vitreous body. Several studies have investigated the role of the complement system in the pathogenesis of glaucoma, especially the role of C3, C1, and complement regulatory proteins. There are studies about experimental autoimmune uveitis and the role of the complement system in the pathogenesis of ocular autoimmune disease. The studies provided the novel finding that complement activation plays a central role in the pathogenesis of ocular autoimmunity and may serve as a potential target for therapeutic intervention. Thus, summing up the results of studies conducted by various authors, we conclude that the complement system has its role in the pathogenesis of various eye pathologies. Identification of the complement system activation as a new direction of local ocular immunity in the pathogenesis of autoimmune uveitis will provide an opportunity for the development of targeted treatment regimens.

Exosomal HSP90 induced by remote ischemic preconditioning alleviates myocardial ischemia/reperfusion injury by inhibiting complement activation and inflammation

Background/AimsThe activation of the complement system and subsequent inflammatory responses are important features of myocardial ischemia/reperfusion (I/R) injury. Exosomes are nanoscale extracellular vesicles that play a significant role in remote ischemic preconditioning (RIPC) cardioprotection. The present study aimed to test whether RIPC-induced plasma exosomes (RIPC-Exo) exert protective effects on myocardial I/R injury by inhibiting complement activation and inflammation and whether exosomal heat shock protein 90 (HSP90) mediates these effects.MethodsRat hearts underwent 30 min of coronary ligation followed by 2 h of reperfusion. Plasma exosomes were isolated from RIPC rats and injected into the infarcted myocardium immediately after ligation. Sixty rats were randomly divided into Sham, I/R, I/R + RIPC-Exo (50 µg/µl), and RIPC-Exo + GA (geldanamycin, 1 mg/kg, administration 30 min before ligation) groups. Cardiomyocyte apoptosis, the release of myocardial markers (LDH, cTnI and CK-MB), infarct size, the expression of HSP90, complement component (C)3, C5a, c-Jun N-terminal kinase (JNK), interleukin (IL)-1β, tumor necrosis factor (TNF)-alpha and intercellular adhesion molecule -1 (ICAM-1) were assessed.ResultsRIPC-Exo treatment significantly reduced I/R-induced cardiomyocyte apoptosis, the release of myocardial markers (LDH, cTnI and CK-MB) and infarct size. These beneficial effects were accompanied by decreased C3 and C5a expression, decreased inflammatory factor levels (IL-1β, TNF-α, and ICAM-1), decreased JNK and Bax, and increased Bcl-2 expression. Meanwhile, the expression of HSP90 in the exosomes from rat plasma increased significantly after RIPC. However, treatment with HSP90 inhibitor GA significantly reversed the cardioprotection of RIPC-Exo, as well as activated complement component, JNK signalling and inflammation, indicating that HSP90 in exosomes isolated from the RIPC was important in mediating the cardioprotective effects during I/R.ConclusionExosomal HSP90 induced by RIPC played a significant role in cardioprotection against I/R injury, and its function was in part linked to the inhibition of the complement system, JNK signalling and local and systemic inflammation, ultimately alleviating I/R-induced myocardial injury and apoptosis by the upregulation of Bcl-2 expression and the downregulation of proapoptotic Bax.

Complement protein levels in serum astrocyte-derived exosomes are associated with cognitive impairment in obstructive sleep apnea.

An association between neuroinflammation and cognitive decline has been established. The complement system regulates neuroinflammation. Dysregulation, impairment, or inadvertent activation of complement components contribute to preclinical Alzheimer's disease. The astrocyte-derived exosome (ADE) complement proteins, including C3b and C5b-9, may be predictive biomarkers of mild cognitive impairment conversion to Alzheimer's disease dementia. We hypothesized that complement proteins might be involved in cognitive impairment during obstructive sleep apnea (OSA). The aim of our study was to explore the correlation between the complement system and mild cognitive impairment (MCI) in patients with OSA. All participants with subjective snoring complaints from the Sleep Medicine Center underwent polysomnography. OSA was defined as apnea-hypopnea index ≥ 5 events/h. MCI was defined as the Montreal Cognitive Assessment < 26 and met the criteria: (1) a subjective cognitive impairment; (2) an objective impairment in 1 or more cognitive domains; (3) complex instrumental daily abilities can be slightly impaired but independent daily living abilities are maintained; and (4) no dementia. The ADEs were isolated immunochemically for enzyme-linked immunosorbent assay quantification of complement proteins, including C3b, C5b-9, and CD55. The participants who received continuous positive airway pressure were followed up and their complement protein levels were reassessed after 1 year of treatment. A total of 212 participants (66.98% males; mean age of 56.71 ± 10.10 years) were divided into the OSA+MCI group (n = 90), OSA-MCI group (n = 79), and controls (normal cognitive state without OSA) (n = 43). The ADE levels of C3b and C5b-9 in the OSA+MCI group were higher than those in the OSA-MCI and control groups. The C3b and C5b-9 were independently associated with cognitive impairment in patients with OSA. The relationship between apnea-hypopnea index and Montreal Cognitive Assessment scores was mediated by C3b and C5b-9. We found no linear correlation between the complement proteins and the severity of OSA. The complement proteins were negatively correlated with global cognitive performance and cognitive subdomains. The complement protein levels significantly decreased after continuous positive airway pressure treatment. Complement proteins were implicated in cognitive impairment in patients with OSA and may be promising biomarkers for predicting cognitive impairment in patients with OSA. Registry: Chinese Clinical Trial Registry; Name: Study on early diagnostic markers in patients with dementia and mild cognitive impairment; URL: https://www.chictr.org.cn/; Identifier: ChiCTR1900021544. Li M, Sun C, Xue S, etal. Complement proteins levels in serum astrocyte-derived exosomes are associated with cognitive impairment in obstructive sleep apnea. J Clin Sleep Med. 2023;19(4):727-739.

Complement component C1q initiates extrinsic coagulation via the receptor for the globular head of C1q in adventitial fibroblasts and vascular smooth muscle cells.

Vascular diseases are highly associated with inflammation and thrombosis. Elucidating links between these two processes may provide a clearer understanding of these diseases, allowing for the design of more effective treatments. The activation of complement component 1 (C1) is a crucial contributor to innate immunity and is associated with significant concentrations of circulating C1q. Many pathological pathways initiate when C1q interacts with gC1qR. This interaction plays a major role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. However, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is the more physiologically relevant coagulation arm, has not been studied. We hypothesized that C1q binding to gC1qR enhances the expression of tissue factor (TF) in adventitial fibroblastsand vascular smooth muscle cells, the primary TF bearing cells in the body. Using an enzyme-linked immunosorbent assay approach, TF expression and the role of gC1qR was observed. Cells were conditioned for 1 hwith C1q or a gC1qR blocker and C1q, to assess the role of gC1qR. Additionally, cell growth characteristics were monitored to assess changes in viability and metabolic activity. Our results indicate that the expression of TF increased significantly after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blockers and C1q exhibited no change in TF expression when compared with cells conditioned with the blocking antibodies alone. Our results show no significant differences in metabolic activity or cell viability under these conditions. This indicates that gC1qR association with C1q induces TF expression and may initiate extrinsic coagulation. Overall, this data illustrates a role for C1q in the activation of extrinsic coagulation and that gC1qR activity may link inflammation and thrombosis.

The Complement System: A Potential Therapeutic Target in Liver Cancer.

Liver cancer is the sixth most common cancer and the fourth most fatal cancer in the world. Immunotherapy has already achieved modest results in the treatment of liver cancer. Meanwhile, the novel and optimal combinatorial strategies need further research. The complement system, which consists of mediators, receptors, cofactors and regulators, acts as the connection between innate and adaptive immunity. Recent studies demonstrate that complement system can influence tumor progression by regulating the tumor microenvironment, tumor cells, and cancer stem cells in liver cancer. Our review concentrates on the potential role of the complement system in cancer treatment, which is a promising strategy for killing tumor cells by the activation of complement components. Conclusions: Our review demonstrates that complement components and regulators might function as biomarkers and therapeutic targets for liver cancer diagnosis and treatment.

C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.

SARS-CoV-2 drives JAK1/2-dependent local complement hyperactivation

Abstract Patients with coronavirus disease 2019 (COVID-19) present a wide range of acute clinical manifestations affecting the lungs, liver, kidneys, and gut. Angiotensin-converting enzyme 2 (ACE2), the best-characterized entry receptor for the disease-causing virus SARS-CoV-2, is highly expressed in the aforementioned tissues. However, the pathways that underlie the disease are still poorly understood. Here, we unexpectedly found that the complement system was one of the intracellular pathways most highly induced by SARS-CoV-2 infection in lung epithelial cells. Infection of respiratory epithelial cells with SARS-CoV-2 generated activated complement component C3a and could be blocked by a cell-permeable inhibitor of complement factor B (CFBi), indicating the presence of an inducible cell-intrinsic C3 convertase in respiratory epithelial cells. Within cells of the bronchoalveolar lavage of patients, distinct signatures of complement activation in myeloid, lymphoid, and epithelial cells tracked with disease severity. Genes induced by SARS-CoV-2 and the drugs that could normalize these genes both implicated the interferon-JAK1/2-STAT1 signaling system and NF-κB as the main drivers of their expression. Ruxolitinib, a JAK1/2 inhibitor, normalized interferon signature genes and all complement gene transcripts induced by SARS-CoV-2 in lung epithelial cell lines but did not affect NF-κB–regulated genes. Ruxolitinib, alone or in combination with the antiviral remdesivir, inhibited C3a protein produced by infected cells. Together, we postulate that combination therapy with JAK inhibitors and drugs that normalize NF-κB signaling could potentially have clinical application for severe COVID-19. This research was financed by the National Heart, Lung, and Blood Institute of the NIH (grant 5K22HL125593 to M. Kazemian; R01HL119215 to J.R.S.); National Institute of General Medical Sciences of the NIH (grant R35GM138283 to M. Kazemian); and Deutsche Forschungsgemeinschaft (fellowship FR3851/2-1 to T. Freiwald) and supported, in part, by the Intramural Research Program of the NIH; the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (project number ZIA/DK075149 to B.A.); the National Heart, Lung, and Blood Institute (NHLBI) (project number ZIA/Hl006223 to C.K.); and the National Institute of Allergy and Infectious Diseases (NIAID) (project number ZIA/AI001175 to M.S.L.). T. Frum is supported by T32DE007057. Funding for part of the work was provided by the University of Michigan Biological Scholars Program (to C.E.W.), LifeARC Charity (to S.K.), and CRUK KHP Centre (to S.K.).

Characterizing F.nucleatumcomplement resistance

Fusobacterium nucleatum (Fn) is a Gram‐negative, anaerobic, oral bacterium that disseminates from the mouth through the bloodstream where it can cause tissue infections, preterm birth, and adverse outcomes in multiple cancers. However, how Fn survives the barrage of immune system attacks while in the bloodstream is wholly unknown. We hypothesized that Fn must be resistant to clearance by the human complement system; a complex network of serum proteins that act to label bacteria and viruses for clearance by immune cells, or directly kill Gram‐negative bacteria by depositing large protein complexes that result in membrane pores and cell death. To test this hypothesis, we incubated Fn with pooled human sera (PHS) with active complement components to determine resistance levels for multiple bacterial strains that are clinically relevant in disease. Here we present varying complement resistance levels in highly similar Fn strains and outline our prediction for the bacterial genes that are driving this immune resistance. These studies have the potential to uncover Fn outer membrane proteins that inactivate the complement system to facilitate their survival and passage throughout the human body. Our goal is to therapeutically target these Fn proteins by disrupting their immune interactions using host‐generated antibodies (vaccine), thereby allowing both the innate and adaptive immune branches to clear these bacteria before they can establish infection and disease.

Activation of Complement Components on Circulating Blood Monocytes From COVID-19 Patients.

The coronavirus disease-2019 (COVID-19) caused by the SARS-CoV-2 virus may vary from asymptomatic to severe infection with multi-organ failure and death. Increased levels of circulating complement biomarkers have been implicated in COVID-19-related hyperinflammation and coagulopathy. We characterized systemic complement activation at a cellular level in 49-patients with COVID-19. We found increases of the classical complement sentinel C1q and the downstream C3 component on circulating blood monocytes from COVID-19 patients when compared to healthy controls (HCs). Interestingly, the cell surface-bound complement inhibitor CD55 was also upregulated in COVID-19 patient monocytes in comparison with HC cells. Monocyte membrane-bound C1q, C3 and CD55 levels were associated with plasma inflammatory markers such as CRP and serum amyloid A during acute infection. Membrane-bounds C1q and C3 remained elevated even after a short recovery period. These results highlight systemic monocyte-associated complement activation over a broad range of COVID-19 disease severities, with a compensatory upregulation of CD55. Further evaluation of complement and its interaction with myeloid cells at the membrane level could improve understanding of its role in COVID-19 pathogenesis.

Pathophysiology of Antiphospholipid Syndrome.

The antiphospholipid syndrome is characterized by antibodies directed against phospholipid-binding proteins and phospholipids attached to cell membrane receptors, mitochondria, oxidized lipoproteins, and activated complement components. When antibodies bind to these complex antigens, cells are activated and the coagulation and complement cascades are triggered, culminating in thrombotic events and pregnancy morbidity that further define the syndrome. The phospholipid-binding proteins most often involved are annexins II and V, β 2 -glycoprotein I, prothrombin, and cardiolipin. A distinguishing feature of the antiphospholipid syndrome is the “lupus anticoagulant.” This is not a single entity but rather a family of antibodies directed against complex antigens consisting of β 2 -glycoprotein I and/or prothrombin bound to an anionic phospholipid. Although these antibodies prolong in vitro clotting times by competing with clotting factors for phospholipid binding sites, they are not associated with clinical bleeding. Rather, they are thrombogenic because they augment thrombin production in vivo by concentrating prothrombin on phospholipid surfaces. Other antiphospholipid antibodies decrease the clot-inhibitory properties of the endothelium and enhance platelet adherence and aggregation. Some are atherogenic because they increase lipid peroxidation by reducing paraoxonase activity, and others impair fetal nutrition by diminishing placental antithrombotic and fibrinolytic activity. This plethora of destructive autoantibodies is currently managed with immunomodulatory agents, but new approaches to treatment might include vaccines against specific autoantigens, blocking the antibodies generated by exposure to cytoplasmic DNA, and selective targeting of aberrant B-cells to reduce or eliminate autoantibody production.

Intravital molecular imaging reveals that ROS-caspase-3-GSDME-induced cell punching enhances humoral immunotherapy targeting intracellular tumor antigens.

Tumor antigens (TAs)-induced humoral immune responses or TAs-specific antibodies have great application prospects for tumor therapy. However, more than half of TAs are intracellular antigens (intra-Ags) that are hardly recognized by antibodies. It is worthy to develop immunotherapeutic strategies for targeting intra-Ags. Methods: We used the far-red fluorescent protein tfRFP as an intracellular antigen to immunize mice and generated a liver metastasis model by injecting tfRFP-expressing B16 melanoma cells (tfRFP-B16) via the spleen. Intravital molecular imaging and atomic force microscopy were performed to visualize the formation of tfRFP antigen-antibody complexes (also known as immune complexes) and punched holes in cell membranes. Results: The results showed that the tfRFP-elicited immune responses inhibited the metastasis of tfRFP-expressing melanoma cells in the liver. In the circulating tfRFP-B16 tumor cells, elevated reactive oxygen species (ROS) induced slight caspase-3 activation, a probable key factor in the cleavage of gasdermin E (GSDME) proteins and punching of holes in the tumor cell membrane. Increased tumor cell membrane permeability led to the release of intra-Ag tfRFP and binding with anti-tfRFP antibodies. The formation of tfRFP antigen-antibody complexes on the membranes of tfRFP-B16 cells activated complement components to form membrane attack complexes to further destroy the cell membrane. Neutrophils were rapidly recruited, and F4/80+ macrophages phagocytized the dying tumor cells. Conclusion: The process of circulating tumor cell elimination in the tfRFP-immunized mice was triggered through the ROS-caspase-3-GSDME pathway to form intra-Ag-antibody immune complexes, which were involved in the activation of the complement system, as well as the recruitment of neutrophils and F4/80+ macrophages. An intra-Ag-elicited humoral immune response is a potent strategy for eliminating liver metastasis, which is unaffected by the liver immune tolerogenic status.

The effect of systemic levels of TNF-alpha and complement pathway activity on outcomes of VEGF inhibition in neovascular AMD

Background/ObjectivesSystemic levels of pro-inflammatory cytokines and activated complement components affect the risk and/or progression of neovascular age-related macular degeneration (AMD). This study investigated the effect of serum pro-inflammatory cytokine levels and complement pathway activity on the clinical response to vascular endothelial growth factor (VEGF) inhibition in neovascular AMD.MethodsSixty-five patients with a new diagnosis of neovascular AMD were observed over a six-month period in a single-centre, longitudinal cohort study. At each visit, the visual acuity score (VAS), central macular thickness (CMT), serum levels of CRP, pro-inflammatory cytokines (TNF-α, IL-1β, IL-2, IL-6 and IL-8), and complement pathway activity were measured. Participant DNA samples were sequenced for six complement pathway single nucleotide polymorphisms (SNPs) associated with AMD.ResultsA statistically significant difference in VAS was observed for serum levels of TNF-α only: there was a gain in VAS (from baseline) of 1.37 for participants below the 1st quartile of mean concentration compared to a reduction of 2.71 for those above the 3rd quartile. Statistical significance was maintained after Bonferroni correction (P value set at <0.006). No significant differences in CMT were observed. In addition, statistically significant differences, maintained after Bonferroni correction, were observed in serum complement activity for participants with the following SNPs: CFH region (rs1061170), SERPING1 (rs2511989) and CFB (rs641153). Serum complement pathway components did not significantly affect VAS.ConclusionsLower serum TNF-α levels were associated with an increase in visual acuity after anti-VEGF therapy. This suggests that targeting pro-inflammatory cytokines may augment treatment for neovascular AMD.

Aberrant activation of the complement system in renal grafts is mediated by cold storage.

Aberrant complement activation leads to tissue damage during kidney transplantation, and it is recognized as an important target for therapeutic intervention. However, it is not clear whether cold storage (CS) triggers the complement pathway in transplanted kidneys. The goal of the present study was to determine the impact of CS on complement activation in renal transplants. Male Lewis and Fischer rats were used, and donor rat kidneys were exposed to 4 h or 18 h of CS followed by transplantation (CS + transplant). To study CS-induced effects, a group with no CS was included in which the kidney was removed and transplanted back to the same rat [autotransplantation (ATx)]. Complement proteins (C3 and C5b-9) were evaluated with Western blot analysis (reducing and nonreducing conditions) and immunostaining. Western blot analysis of renal extracts or serum indicated that the levels of C3 and C5b-9 increased after CS + transplant compared with ATx. Quite strikingly, intracellular C3 was profoundly elevated within renal tubules after CS + transplant but was absent in sham or ATx groups, which showed only extratubular C3. Similarly, C5b-9 immunofluorescence staining of renal sections showed an increase in C5b-9 deposits in kidneys after CS + transplant. Real-time PCR (SYBR green) showed increased expression of CD11b and CD11c, components of complement receptors 3 and 4, respectively, as well as inflammatory markers such as TNF-α. In addition, recombinant TNF-α significantly increased C3 levels in renal cells. Collectively, these results demonstrate that CS mediates aberrant activation of the complement system in renal grafts following transplantation.NEW & NOTEWORTHY This study highlights cold storage-mediated aberrant activation of complement components in renal allografts following transplantation. Specifically, the results demonstrate, for the first time, that cold storage functions in exacerbation of C5b-9, a terminal cytolytic membrane attack complex, in renal grafts following transplantation. In addition, the results indicated that cold storage induces local C3 biogenesis in renal proximal cells/tubules and that TNF-α promotes C3 biogenesis and activation in renal proximal tubular cells.

Complement Component C1q Initiates Extrinsic Coagulation via gC1qR

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. When C1q interacts with gC1qR, many pathological pathways initiate and C1q/gC1qR plays a role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. While this link between inflammation and thrombosis is important, the intrinsic cascade plays a minor role in physiological and pathological thrombosis. The effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is more physiologically relevant, has not been studied. Extrinsic coagulation initiates upon vessel damage, exposing tissue factor (TF) to the blood. Adventitial fibroblasts and vascular smooth muscle cells are the primary TF bearing cells. We hypothesized that C1q binding to gC1qR enhances the expression of sub-endothelial TF in these cells. The objective of this study was to elucidate the role of gC1qR-mediated TF expression. Using a solid-phase ELISA, TF expression was observed on aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti-gC1qR (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. Further, a capture ELISA was used to assess released TF under these same conditions. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 35% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies exhibited a marked decrease in TF expression (by approximately 10-20%) when compared to cells conditioned with C1q alone. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying common inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF can support extrinsic coagulation activity.